TW201900212A - Method for treating immune-related adverse events in cancer therapy using soluble CD24 - Google Patents

Abstract

The present invention relates to the use of a CD24 protein for treating immune-related adverse events (irAEs) associated with cancer immunotherapy.

Description

Method for treating immune-related adverse events in cancer therapy using soluble CD24

The present invention relates to the use of CD24 protein for the treatment of immune-related adverse events (irAEs) associated with cancer immunotherapy.

The immune system has the ability to identify and eliminate experimental model systems and patients' cancers. Therefore, cancer immunotherapy is emerging as one of the most promising areas of cancer therapy. Active cancer immunotherapy involves agents that amplify the natural immune response (including antibodies against PD-1, PD-L1, or CTLA-4); small molecules that regulate the tumor microenvironment; or the use of in vitro stimulation of tumor infiltrating lymphocytes ( TILs), activated natural killer (NK) cells, or genetically engineered T cells (T-cells modified with chimeric antigen receptors [CARs] and T cell receptors [TCR]) and acceptor cell transfer (ACT). Alternatively, other cancer immunotherapies that directly target tumors can indirectly activate the immune system (including antibodies that target cancer, such as anti-Her-2 antibodies in the case of solid cancer, anti-CD20 antibodies in the case of malignant B-cell disease, or Anti-GM2 antibody in the case of neuroblastoma). Antibodies against PD-1, PD-L1, and CTLA-4 and tumor-targeting antibodies have shown substantial benefits in the treatment of solid tumors and hematological tumors. Receptive cell immunotherapy, such as CAR T cells, has shown impressive effects against hematological tumors, such as leukemia, but has only limited effects on solid tumors. Although CAR-T immunotherapy has more targeted hematological malignancies, T-cell immunotherapy using genetically modified TCR has more targeted solid tumors.

Tumors avoid immune elimination by creating highly tolerated and immunosuppressive tumor microenvironments (TMEs). Therefore, one of the important goals of immuno-oncology is to understand the variable tolerance mechanisms utilized by tumors in order to exclude such mechanisms that allow tumor infiltration and activation, thereby allowing the immune system to destroy tumor cells. Despite its therapeutic promise, systemic delivery of immunotherapy can also break through self-tolerance. This in turn causes mild to severe inflammatory reactions in a variety of organ systems and, in some cases, life-threatening autoimmunity, commonly referred to as immune-related adverse events (irAEs). As immunotherapeutic methods are being expanded with more and more effective combinations to address more cancer indications, controlling non-specific irAEs has become a key goal for these next-generation cancer immunotherapies.

Treatment with antibodies against PD-1, PD-L1 and CTLA-4 has been shown to be a powerful tool to enhance the anti-tumor immunity of preclinical models. Monotherapy using antibodies against CTLA4 promotes rejection of transplantable tumors of different origin. Based on promising preclinical tumor model studies, the clinical potential of antibodies against CTLA4 in different human malignant diseases has been explored. Although anti-CTLA4 (Ipilimumab, commercially available as Yervoy and disclosed in US Patent No. 6,984,720) has shown efficacy in treating melanoma, the treatment and targeting of CTLA4 is associated with autoimmune-like toxicity. Anti-PD-1 and anti-PD-L1 antibodies display significantly higher clinical response and significantly lower irAE. However, severe autoimmune adverse events still occur in about 15-20% of cancer patients. In addition, anti-CTLA4 mAbs, such as Ipilimumab and Tremelimumab, are used in combination therapy with anti-PD-1 / PD-L1 antibodies that have excellent therapeutic effects. However, the improved treatment effect is even associated with higher grade 3 and 4 organ toxicity rates. IrAEs have been reported in a number of body parts, including the gastrointestinal tract, skin, kidney, pancreas, liver, and central and peripheral nervous systems. The incidence and severity of irAEs correlates with overall patient response and survival for checkpoint blockades, suggesting that both antitumor and autoimmune responses are sensitive to checkpoint immunotherapy. Such irAEs can be broadly classified as "autoimmune toxicity" or the so-called "medium target, non-tumor toxicity", which is caused by the antigen produced on the host tissue when the targeted tumor-associated antigen is expressed in non-malignant tissue. Caused by specific attacks. Following genetically engineered T-cell infusion targeting MAGE-A3, autoimmune toxicity has caused lethal toxicity.

Genetically engineered T-cell therapies, such as CAR-T, are also associated with irAEs that limit their use, but here, adverse events are more often cytokine-related toxicity. CAR-T cells cause the expansion of T cells in vivo, which can cause the release of cytotoxic levels. This systemic inflammatory response is individually referred to as the interleukin storm or the interleukin release syndrome (CRS). Infusion reactions are also a common phenomenon of antibodies and Fc fusion protein therapeutics, and are associated with symptoms in the following ranges: mild nausea and fever, to life-threatening multiple organ failure. Active support for care is necessary for all patients experiencing CAR T cell toxicity, of which early intervention for hypotension and treatment of concurrent infections are essential. However, pharmacological management is complicated by immunosuppressive therapies that eliminate the risk of CAR T cell anti-malignant disease activity and is the main reason why prophylactic immunosuppression is not used. Tocilizumab to block interleukin-6 receptor is still the main pharmacological therapy for CRS, but the indications for administration vary depending on the treatment center.

CRS has also been accompanied by other forms of cancer therapy, which are related to the rapid lysis of malignant cells, leading to an acute allergic reaction or a phenomenon known as tumor lysis syndrome (TLS). Because cytolysis can cause the release of intracellular components, called dangerous (damage) related molecular patterns (DAMPs), if not properly controlled, it can cause inflammation and create conditions for autoimmune diseases. However, traditional immunosuppressants can be problematic due to adverse events in cancer therapy, as all forms of cancer therapy believe that anti-cancer immune responses are directly or indirectly required.

Therefore, there is a large unmet need in medicine to treat irAEs while maintaining cancer immunity.

This article provides a method of treating, alleviating, minimizing or preventing immune-related adverse events (irAEs) associated with cancer immunotherapy by administering CD24 protein to individuals in need. irAE may be diarrhea or another gastrointestinal disorder, pure red blood cell hypoplasia, small red blood cell anemia, lupus, autoimmune nephritis, autoimmune hepatitis, pneumonia, myocarditis, pericarditis, endocrine disease, Addison's disease ), Sjogren's syndrome or type I diabetes. The CD24 protein may comprise mature human CD24 or a variant thereof. The sequence of mature human CD24 may comprise the amino acid sequence set forth in SEQ ID NO: 1 or 2. The CD24 protein may contain any or all of the extracellular domains of human CD24. The sequence of the CD24 protein may comprise a signal sequence having the amino acid sequence set forth in SEQ ID NO: 4 to allow secretion from a cell expressing the protein. The signal peptide sequence may be a sequence found on other transmembrane or secreted proteins, or modified sequences of existing signal peptides known in the art. The CD24 protein may be soluble and / or may be glycosylated. The CD24 protein can be produced using a eukaryotic protein expression system, which can include a vector contained in a Chinese hamster ovary cell line or a replication-deficient retrovirus vector. Replication-deficient retroviral vectors can be stably integrated into the genome of eukaryotic cells.

The CD24 protein can include a protein tag that can be fused at the N- or C-terminus of the CD24 protein. The protein may comprise a portion of a mammalian immunoglobulin (Ig), which may be the Fc region of a human Ig protein. The human Ig protein may include a hinge region and CH2 and CH3 domains of the human Ig protein, and the human Ig protein may be IgG1, IgG2, IgG3, IgG4, or IgA. The Fc region may also include the hinge region of IgM and the CH2, CH3, and CH4 domains. The CD24 protein may comprise an amino acid sequence set forth in SEQ ID NO: 5, 6, 8, 9, 11, or 12.

The cancer immunotherapy may be an anti-CTLA4 antibody, and the antibody may be ipilimumab. Anti-CTLA4 antibodies can be administered in combination with another therapy. Cancer therapy can also be an anti-PD-1 antibody, which can be administered in combination with another therapy. Cancer therapy can also be an anti-PD-L1 antibody, which can be administered in combination with another therapy. Cancer therapy can also be chimeric antigen T cells, T cells modified with T cell receptors, or activated natural killer cells. Cancer therapy can also be radiation therapy, chemotherapy, or cancer therapy involving antibodies that target cancer cells.

Also described herein is a method of treating, reducing or preventing GvHD by administering CD24 protein to an individual in need, who may receive an allogeneic hematopoietic stem cell transplant (HSCT) Have received or are receiving activated natural killer (aNK) cells.

Further described herein is a method of preventing or treating irAEs associated with massive tumor lysis that occurs during cancer treatment by administering CD24 to individuals in need. Cancer therapy can be radiation therapy, chemotherapy, or anti-cancer antibodies that allow cancer cells to be killed directly.

The inventors have surprisingly discovered that the soluble form of CD24 is highly effective in treating immune-related adverse events (irAEs). The effect can be mediated via DAMPs. Pattern recognition involves inflammatory responses triggered by pathogen-associated molecular patterns and tissue damage-related molecular patterns (referred to as PAMPs and DAMPs, respectively). The inventors have realized that recent studies have shown that an increased host response to DAMPs may play a part in the pathogenesis of inflammation and autoimmune diseases. DAMPs were found to promote the production of inflammatory cytokines and autoimmune diseases in animal models, and therefore DAMPs inhibitors such as HMGB1 and HSP90 were found to improve rheumatoid arthritis (RA) (4-6). TLRs, RAGE-R, DNGR (encoded by Clec9A), and macrophage-induced type C lectin (Mincle) have been shown to be receptors responsible for mediating inflammation initiated by various DAMPs (2, 7-14).

Recent works by the inventors show that the CD24-sialic acid-binding immunoglobulin-like lectin G interaction makes innate immunity against DAMPs different from innate immunity against PAMPs (15, 16). Sialic acid-binding immunoglobulin-like lectin proteins are members of a variety of membrane-associated immunoglobulin (Ig) superfamilies that recognize sialic acid-containing structures. Most sialic acid-binding immunoglobulin-like lectins have an intracellular immune tyrosine inhibitory motif (ITIM) that binds to SHP-1, SHP-2, and Cbl-b, a key regulator of the inflammatory response . The present inventors have reported that CD24 is the first natural of sialic acid-binding immunoglobulin-like lectin (sialic acid-binding immunoglobulin-like lectin G in mice and sialic acid-binding immunoglobulin-like lectin 10 in humans) Ligand (15). Sialic acid-binding immunoglobulin-like lectin G interacts with sialylated CD24 to inhibit TLR-mediated host responses to DAMPs (such as HMGB1) via the SHP-1 / 2 signaling mechanism (15).

Human CD24 is a small molecule anchored by GPI, which is encoded by an open reading frame of 240 base pairs in the CD24 gene (28). Of the 80 amino acids, the first 26 constitute the signal peptide, while the last 23 serve as cleavage signals to achieve GPI tail ligation. Therefore, mature human CD24 molecules have only 31 amino acids. One of the 31 amino acids has polymorphisms among human populations. The C-to-T conversion at nucleotide 170 of the open reading frame causes alanine (a) to be replaced by valine (v). Since this residue is located at the N-terminus nearest to the cleavage site, and because the substitution is not conservative, the two dual genes can be expressed on the cell surface with different efficiency. Indeed, cDNA transfection studies have demonstrated that the CD24 ^v dual gene appears more efficiently on the cell surface (28). Based on this, CD24 ^{v / v} PBL showed higher amounts of CD24, especially on T cells.

The inventors have demonstrated that CD24 negatively regulates the host response to cellular DAMPs that are released as a result of tissue or organ damage, and at least two overlapping mechanisms can explain this activity. First, CD24 binds to several DAMPs, including HSP70, HSP90, HMGB1, and nucleolin, and inhibits the host response to these DAMPs. For this reason, it is assumed that CD24 can retain inflammatory stimuli to prevent interaction with its receptor TLR or RAGE. Secondly, using a mouse model of acetaminophen-induced liver necrosis and ensuring inflammation, the inventors have demonstrated that CD24 is responsible for tissue damage through interactions with its receptor, sialic acid-binding immunoglobulin-like lectin G. The host response provides powerful negative regulation. To obtain this activity, CD24 can bind to sialic acid-binding immunoglobulin-like lectin G and stimulate signal transduction by sialic acid-binding immunoglobulin-like lectin G, in which sialic acid-binding immunoglobulin-like lectin G binds to SHP1 to trigger negative向 调整。 To adjust. Both mechanisms work synergistically, as mice with targeted mutations in either gene produce a much stronger inflammatory response. In fact, DCs obtained from bone marrow culture of CD24-/-or sialic acid-binding immunoglobulin-like lectin G-/-mice produce higher amounts of inflammatory cytokines when stimulated with HMGB1, HSP70 or HSP90. To the inventors' knowledge, CD24 is the only inhibitory DAMP receptor capable of stopping DAMPs from triggering inflammation, and drugs that specifically target the host's inflammatory response to tissue damage are currently unavailable. In addition, the inventors have used mouse models of RA, MS, and GvHD to demonstrate that exogenous soluble CD24 protein can alleviate DAMP-mediated autoimmune diseases. 1. Definitions.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, as used in the description and the scope of the accompanying patent application, the singular forms "a / an" and "the" include plural referents.

For the purposes of this description of numerical ranges, each intermediate value with the same accuracy is expressly covered. For example, the range 6-9 covers the values 7 and 8 in addition to 6 and 9, and the range 6.0-7.0 explicitly covers the values 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0.

A "peptide" or "polypeptide" is a linked amino acid sequence and may be natural, synthetic, or a modification or combination of natural and synthetic.

"Substantially identical" may mean that the first and second amino acid sequences are at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 amino acids in at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% are consistent.

When referring to preventing disease in an animal, "treatment or treating" means preventing, suppressing, suppressing or completely eliminating the disease. Preventing disease involves administering a composition of the invention to an animal before the onset of the disease. Inhibiting a disease involves administering a composition of the invention to an animal after its induction, but before its clinical manifestation. Inhibiting a disease involves administering a composition of the invention to an animal prior to the clinical manifestation of the disease.

A "variant" may mean a peptide or polypeptide that differs in amino acid sequence due to amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity. Representative examples of "biological activity" include the ability to bind to toll-like receptors and to be bound by specific antibodies. A variant may also mean a protein whose amino acid sequence is substantially identical to the amino acid sequence of a reference protein that maintains at least one biological activity. Conservative substitutions of amino acids (ie, replacement of amino acids by different amino acids having similar properties, such as the degree of hydrophilicity and distribution of charged regions) are generally recognized in the art as involving typically small changes. As understood in the art, these minor changes can be identified in part by considering the hydrophilic index of amino acids. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydrophilic index of amino acids is based on their hydrophobicity and charge considerations. It is known in the art that amino acids with similar hydrophilic indices can be substituted and still retain protein function. In one aspect, an amino acid having a hydrophilic index of ± 2 is substituted. The hydrophilic nature of amino acids can also be used to demonstrate substitutions that allow proteins to maintain biological functions. Considering the hydrophilicity of amino acids in the case of peptides allows calculation of the maximum local average hydrophilicity of that peptide, which is a suitable measure that has been reported to be sufficiently correlated with antigenicity and immunogenicity. US Patent No. 4,554,101, which is fully incorporated herein by reference. As understood in the art, substitution of amino acids with similar hydrophilic values can produce peptides that retain biological activity (eg, immunogenicity). Substitutions can be made with amino acids having hydrophilic values within ± 2 of each other. The hydrophobic index and hydrophilic value of amino acids are both affected by the specific side chains of the amino acids. According to his observations, it should be understood that amino acid substitution compatible with biological functions depends on the relative similarity of amino acids. In particular, the relative similarity of the side chains of their amino acids, such as hydrophobicity, hydrophilicity, etc. Performance, charge, size and other characteristics. CD24

Provided herein is a CD24 protein, which may comprise mature CD24 or a variant thereof. Mature CD24 corresponds to the extracellular domain (ECD) of CD24. Mature CD24 can be from a human or another mammal. As described above, the mature human CD24 protein has 31 amino acid lengths and has variable alanine (A) or valine (V) residues at its C-terminus: SETTTGTSSNSSQSTSNSGLAPNPTNATTK (V / A) (SEQ ID NO : 1)

C-terminal valine or alanine may be immunogenic and can be omitted from the CD24 protein, thereby reducing its immunogenicity. Therefore, the CD24 protein may contain a human CD24 amino acid sequence lacking a C-terminal amino acid: SETTTGTSSNSSQSTSNSGLAPNPTNATTK (SEQ ID NO: 2)

Despite considerable sequence changes in the amino acid sequence of mature CD24 protein from mice and humans, it is a functional equivalent because human CD24Fc has been shown to be active in mice. The amino acid sequence of human CD24 ECD showed some sequence conservation with mouse proteins (39% identity; gene bank accession number NP_033976). Not surprisingly, however, since the CD24 ECD is only 27-31 amino acids (depending on the species), the percent identity will not be higher, and it binds to some of its receptors, such as sialic acid binding Immunoglobulin-like lectin (10 / G) is mediated by its sialic acid and / or galactose sugars of glycoproteins. The extracellular domain of human sialic acid-binding immunoglobulin-like lectin-10 (Gene Bank Accession No. AF310233) and its murine homolog sialic acid-binding immunoglobulin-like lectin-G (Gene Bank Accession No. NP_766488) receptor protein The amino acid sequence identity was 63% (Figure 2). Because the sequence conservation between mouse and human CD24 mainly exists at the C-terminus and a large number of glycosylation sites, CD24 protein can tolerate significant variations of mature CD24 protein, especially their mutations do not affect the conservation of C-terminus. The residues did not affect the glycosylation site of mouse or human CD24. Therefore, the CD24 protein may contain the amino acid sequence of mature murine CD24: NQTSVAPFPGNQNISASPNPTNATTRG (SEQ ID NO: 3).

The amino acid sequence of human CD24 ECD showed sequence conservation (52% identity; UniProt Registration Number UniProtKB-I7GKK1) with the presence of cynomolgus monkey protein, which is greater than that of mice. Again, this is not surprising given the fact that since ECD has only 29-31 amino acid lengths in these species, the percent identity will not be higher, and the role of sugar residues is to bind to them Receptor. The amino acid sequence of cynomolgus sialic acid-binding immunoglobulin-like lectin-10 receptor has not been determined, but between humans and rhesus sialic acid-binding immunoglobulin-like lectin-10 (Gene Bank Accession No. XP_001116352) protein The amino acid sequence identity was 89%. Therefore, the CD24 protein may also contain the amino acid sequence of mature cynomolgus monkey (or rhesus) CD24: TVTTSAPLSSNSPQNTSTTPNPANTTTKA (SEQ ID NO: 10)

The CD24 protein may be soluble. The CD24 protein may further comprise an N-terminal signal peptide to allow secretion from cells expressing the protein. The signal peptide sequence may include the amino acid sequence MGRAMVARLGLGLLLLLLALLLPTQIYS (SEQ ID NO: 4). Alternatively, the signal sequence may be any one of other signal sequences found on other transmembrane or secreted proteins or modified on existing signal peptides known in the art. a. fusion

The CD24 protein can be fused at its N- or C-terminus to a protein tag, which can include a portion of a mammalian Ig protein that can be from human or mouse or another species. The portion may comprise an Fc region of the Ig protein. The Fc region may include at least one of a hinge region, a CH2, a CH3, and a CH4 domain of the Ig protein. The Ig protein can be human IgG1, IgG2, IgG3, IgG4, or IgA, and the Fc region can include the hinge region of Ig and the CH2 and CH3 domains. The Fc region may contain the human immunoglobulin G1 (IgG1) isotype (SEQ ID NO: 7). The Ig protein can also be IgM, and the Fc region can include the hinge region of IgM and the CH2, CH3, and CH4 domains. The protein tag may be an affinity tag that facilitates protein purification, and / or a solubility enhancing tag that enhances the solubility and recovery of functional proteins. Protein tags can also increase the value of CD24 protein. The protein tag may also include GST, His, FLAG, Myc, MBP, NusA, thioredoxin (TRX), small ubiquitin-like modulator (SUMO), ubiquitin (Ub), albumin, or camelid Ig. Methods for preparing and purifying fusion proteins are well known in the art.

According to preclinical studies, in order to construct the fusion protein CD24Fc identified in the example, a truncated form of 30 native amino acids of CD24 has been used, which lacks the last polymorphic amino acid (ie, the GPI signal cleavage site) , Mature CD24 protein with SEQ ID NO: 2). The mature human CD24 sequence was fused to the human IgG1 Fc domain (SEQ ID NO: 7). The full-length CD24Fc fusion protein is provided in SEQ ID NO: 5 (FIG. 1), and a processed form of the CD24Fc fusion protein secreted by the cell (ie, a signal sequence lacking division) is provided in SEQ ID NO: 6. A fusion of a processed polymorphic variant of mature CD24 (ie, the mature CD24 protein with SEQ ID NO: 1) and an IgG1 Fc may comprise SEQ ID NO: 11 or 12. b. produce

CD24 proteins can be largely glycosylated and can be involved in CD24 functions, such as co-stimulation of immune cells and interaction with damage-associated molecular model molecules (DAMP). CD24 proteins can be prepared using a eukaryotic expression system. The performance system may require the vector to be expressed in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. The system can also be a viral vector, such as a replication-deficient retroviral vector that can be used to infect eukaryotic cells. The CD24 protein can also be produced by a stable cell line that expresses the CD24 protein via a vector or part of a vector that has been integrated into the cell's genome. Stable cell lines can express CD24 protein via an integrated replication-deficient retroviral vector. The performance system can be GPEx ™. c. pharmaceutical composition

The CD24 protein may be included in a pharmaceutical composition, which may include a CD24 protein in a pharmaceutically acceptable amount. The pharmaceutical composition may include a pharmaceutically acceptable carrier. The pharmaceutical composition may include a solvent that stabilizes the CD24 protein for an extended period of time. The solvent may be PBS, which can stabilize the CD24 protein at -20 ° C (-15 to -25 ° C) for at least 66 months. The solvent is able to hold the CD24 protein in combination with another drug.

The pharmaceutical composition can be formulated for parenteral administration, including (but not limited to) injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulations including, but not limited to, suspending, stabilizing and dispersing agents. The composition may also be provided in powder form for reconstitution with a suitable vehicle including, but not limited to, sterile pyrogen-free water.

The pharmaceutical composition can also be formulated as a reservoir formulation, which can be administered by implantation or by intramuscular injection. The composition can be formulated with a suitable polymer or hydrophobic substance (such as an emulsion in an acceptable oil), an ion exchange resin, or a sparingly soluble derivative (eg, a sparingly soluble salt). Subcutaneous injection formulations may be particularly relevant for indications such as lupus and its related manifestations and complications. d. dosage

The CD24 protein dosage can ultimately be determined through clinical trials to determine dosages with acceptable toxicity and clinical efficacy. The initial clinical dose can be estimated from pharmacokinetic and toxicity studies in rodents and non-human primates. The CD24 protein dosage may be from 0.01 mg / kg to 1000 mg / kg, and may be from 1 to 500 mg / kg, depending on the desired effect and the route of administration. CD24 protein can be administered by intravenous infusion or subcutaneous, intramural (i.e., cavity or organ wall) or intraperitoneal injection, and the dose can be 10-1000 mg, 10-500 mg, 10-240 mg , 10-120 mg, or 10, 30, 60, 120, or 240 mg, of which the individual is a human. 3. Treatment a. Immune-related adverse events

Provided herein is a method for alleviating, reducing, minimizing or treating irAEs by administering CD24 protein to individuals in need. irAEs may be related to cancer therapy, and individuals may be cancer patients. Cancer therapy may be cancer immunotherapy. The CD24 protein can be administered to individuals who are at or at risk of irAEs associated with cancer therapy. CD24 protein can be used prophylactically to prevent irAEs before cancer therapy begins or before clinical signs of irAEs become apparent. After initiating cancer therapy and diagnosing clinical symptoms, CD24 protein can also be therapeutically administered to treat irAEs. irAE can be diarrhea or another gastrointestinal disorder, pure red blood cell hypoplasia, microcytic anemia, lupus, autoimmune nephritis, autoimmune hepatitis, pneumonia, myocarditis, pericarditis, endocrine disease, Addison's disease, hypogonadism Disease, Hugh's syndrome, or type I diabetes.

Cancer therapy can be active immunotherapy. Examples of active immunotherapy include anti-CTLA4, anti-PD-1, anti-PD-L1, anti-TNF, antibodies against another TNF receptor family member, anti-LAG3, anti-TIM3, and small or macromolecular inhibition of the tumor microenvironment Agent. In particular, the cancer therapy may be an anti-CTLA4 immunotherapy, and the CD24 protein may be combined with an anti-CTLA4 immunotherapy or administered to an individual in the context of an anti-CTLA4 immunotherapy. Examples of anti-CTLA4 antibodies include Yervoy and Tremilimumab.

In another embodiment, the cancer therapy may be an anti-PD-1 / PD-L1 immunotherapy, which may be an anti-PD-1 antibody or an anti-PD-L1 antibody. Examples of anti-PD-1 antibodies include nivolumab (Opdivo-Bristol Myers Squibb) and Pembrolizumab (Keytruda, MK-3475, Merck). Examples of anti-PD-L1 antibodies include Atezolizumab (Tecentriq, Roche), Avelumab (Merck KGaA and Pfizer), and durvalumab (Imfinzi, Astra- Zeneca).

In yet another embodiment, the cancer therapy may be a combination therapy comprising anti-CTLA-4 and anti-PD-1 monoclonal antibodies (mAbs). The combination of anti-CTLA-4 and anti-PD-1 has emerged as the strongest and most durable cancer immunotherapy. However, the autoimmune effects associated with combination therapy are very serious, with more than 50% of melanoma patients experiencing grade 3 and 4 organ toxicity. Therefore, the main challenge of cancer immunotherapy is how to reduce the adverse effects of combination therapies without affecting the therapeutic efficacy. Among the two components of the combination therapy, anti-CTLA-4 mAb exhibited significantly more immunotherapy-related adverse effects (irAE).

Cancer therapies can be adaptive cell transfer (ACT), which uses tumor infiltrating lymphocytes (TILs) stimulated in vitro or genetically engineered T cells (chimeric antigen receptors [CARs] or T cell receptors [ TCR] modified T cells). In particular, by promoting the rapid death of normal cells and cancer cells, cancer therapies such as CAR-T cells can promote the release of damage-associated molecular patterns (DAMPs) and thus cause the cytokine release syndrome, which can also cause extensive organ function obstacle. Therefore, the CD24 protein can be used to reduce or neutralize the effects of DAMPs and to alleviate, minimize or treat the interleukin storm caused. CAR-T cells can damage normal cells if they target tumor-associated antigens that are also expressed on non-tumor cells and tissues. CAR-T therapy can be used to treat hematological tumors such as acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia, adult myeloid leukemia (AML), and diffuse large B-cell lymphoma (DLBCL) , Non-Hodgkin Lymphoma (NHL), chronic lymphocytic leukemia (CLL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), and multiple myeloma ( MM). Examples of CAR-T therapies include targeting B-cell surface antigens CD19 (such as JCAR017 and JCAR014 [Juno Therapeutics]), CTL019 (tisagenlecleucel-T) [Novartis], and KTE-C19 [Cicero ( axicabtagene ciloleucel), Kite Pharma]), and CD22 (JCAR014 [Juno Therapeutics]). Other examples of CAR-T therapy include targeted therapies for L1-CAM (JCAR023 [Juno Therapeutics]), ROR-1 (JCAR024 [Juno Therapeutics]), and MUC16 (JCAR020 [Juno Therapeutics]). Examples that target TCR-modified T cells include examples that target MAGE-A3, such as KITE-718 (Kite Pharma); targets that target Wilms tumor antigen 1 (WT-1) Examples, such as JTCR016 (Juno Therapeutics); and NY-ESO-1.

Cancer therapies can be therapies that involve the rapid killing of cancer cells, such as radiation and chemotherapy. The resulting tumor lysis can cause the release of DAMPs, which initiates the inflammatory cascade. Such indications would be particularly suited to preventive treatment of CD24 protein. b. graft versus host disease

This article also provides a method for reducing or treating GvHD in a subject by administering CD24 protein to a subject in need, who may have received or is undergoing allogeneic hematopoietic stem cell transplantation (HSCT) Receive activated natural killer (aNK) cells. NK cells can enhance transplantation and mediate graft anti-leukemia after allogeneic HSCT, but the efficacy of graft-mediated leukemic anti-leukemia after HSCT is naturally limited. Preclinical studies have demonstrated that activation of NK cells up-regulates the expression of activated receptors and enhances killing capacity (Shah et al., 2015). This was then tested in a clinical trial that transplanted donor-derived donor-derived T cells after depleting non-myeloablative peripheral blood stem cells to children and young adults with high-risk solid tumors. The permissive metastasis of activated NK cells (aNK-DLI) has been studied. aNK-DLI exhibits potent killing ability and shows high levels of activated receptor performance. However, 5 of the 9 graft recipients experienced acute graft-versus-host disease (GVHD) after aNK-DLI, with Grade 4 GVHD observed in 3 individuals. GVHD was more common among matched unrelated donors than matched donor sibling recipients, and was associated with higher donor CD3 chimeric traits. Given that T-cell doses are below the threshold required for GVHD in this context, it is inferred that aNK-DLI causes the observed acute GVHD, which may be achieved by enhancing the potential T-cell allograft reactivity. c. administration

The administration route of the pharmaceutical composition may be parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, intraarticular, and direct injection. The pharmaceutical composition can be administered to a human patient, cat, dog, or large animal. The composition can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 times per day. d. Combination therapy

The CD24 protein can be used in combination with another agent to further reduce, alleviate or treat the interleukin release syndrome (CRS). Interleukin release syndrome is associated with increased levels of circulating interleukins, including interleukin (IL) -6 and IFN-γ. Therefore, with or without corticosteroids, other agents may be tocilizumab (Actemra), anti-IL-6 receptor antibodies, or another cytokines targeting agent, which can be used for immunosuppression can Used to reverse symptoms, especially in patients receiving CAR-T. Other agents used in CRS management can be siltuximab (anti-IL-6, Sylvant), etanercept (TNFα inhibitor, Enbrel), infliximab (anti-TNFα, Remicade ), Or anakinra (interleukin-1 receptor antagonist, Kineret). CD24 protein can be used to target and alleviate the effects of DAMPs released from damaged tissues and initiate an inflammatory cascade, while combination therapy can target effector interleukin molecules, thereby providing a two-pronged complementary approach to alleviate, reduce or Treatment of CRS.

CD24 protein can be administered simultaneously or rhythmically with other therapies. As used herein, the term "simultaneously" means that the CD24 protein and another therapy are administered within 48 hours of each other, preferably 24 hours, more preferably 12 hours, yet more preferably 6 hours, and most preferably 3 hours or Less than 3 hours. As used herein, the term "rhythmically" means that the agent is administered with another therapy at a different time and at a specific frequency (as opposed to repeated administration).

CD24 protein can be administered at any point before another therapy, including about 120 hours, 118 hours, 116 hours, 114 hours, 112 hours, 110 hours, 108 hours, 106 hours, 104 hours, 102 hours, 100 hours, 98 Hours, 96 hours, 94 hours, 92 hours, 90 hours, 88 hours, 86 hours, 84 hours, 82 hours, 80 hours, 78 hours, 76 hours, 74 hours, 72 hours, 70 hours, 68 hours, 66 hours, 64 hours, 62 hours, 60 hours, 58 hours, 56 hours, 54 hours, 52 hours, 50 hours, 48 hours, 46 hours, 44 hours, 42 hours, 40 hours, 38 hours, 36 hours, 34 hours, 32 hours , 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 Hours, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes and 1 minute. The CD24 protein can be administered at any time before the second therapy of the CD24 protein, including about 120 hours, 118 hours, 116 hours, 114 hours, 112 hours, 110 hours, 108 hours, 106 hours, 104 hours, 102 hours, 100 Hours, 98 hours, 96 hours, 94 hours, 92 hours, 90 hours, 88 hours, 86 hours, 84 hours, 82 hours, 80 hours, 78 hours, 76 hours, 74 hours, 72 hours, 70 hours, 68 hours, 66 hours, 64 hours, 62 hours, 60 hours, 58 hours, 56 hours, 54 hours, 52 hours, 50 hours, 48 hours, 46 hours, 44 hours, 42 hours, 40 hours, 38 hours, 36 hours, 34 hours , 32 hours, 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 Hours, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, and 1 minute.

CD24 protein can be administered at any time after another therapy, including about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 Minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours , 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 Hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours and 120 hours. CD24 protein can be administered at any point after the previous CD24 therapy, including approximately 120 hours, 118 hours, 116 hours, 114 hours, 112 hours, 110 hours, 108 hours, 106 hours, 104 hours, 102 hours, 100 hours, 98 Hours, 96 hours, 94 hours, 92 hours, 90 hours, 88 hours, 86 hours, 84 hours, 82 hours, 80 hours, 78 hours, 76 hours, 74 hours, 72 hours, 70 hours, 68 hours, 66 hours, 64 hours, 62 hours, 60 hours, 58 hours, 56 hours, 54 hours, 52 hours, 50 hours, 48 hours, 46 hours, 44 hours, 42 hours, 40 hours, 38 hours, 36 hours, 34 hours, 32 hours , 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 Hours, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes and 1 minute. Example 1 Pharmacokinetics of CD24 in mice

1 mg of CD24Fc (CD24Fc) was injected into untreated C57BL / 6 mice and 3 mice were collected at different time points (5 minutes, 1 hour, 4 hours, 24 hours, 48 hours, 7 days, 14 days, and 21 days) Blood samples from mice at each time point. CD24Fc was detected by diluting serum 1: 100 and using sandwich ELISA, using purified anti-human CD24 (3.3 μg / ml) as capture antibody and peroxidase-bound goat anti-human IgG Fc (5 μg / ml) as detection antibody. content. As shown in Figure 3A, the decay curve of CD24Fc revealed that the protein exhibited typical biphasic decay. The first biodistribution phase has a half-life of 12.4 hours. The second phase follows a model of primary elimination from the central compartment. The half-life of the second phase is 9.54 days, which is similar to the half-life of antibodies in vivo. These data indicate that the fusion protein is very stable in the bloodstream. In another study where the fusion protein was injected subcutaneously, an almost identical half-life of 9.52 days was observed (Figure 3B). More importantly, although it took about 48 hours for the CD24Fc to reach its peak level in the blood, the total amount of fusion protein caused by any injection route was substantially the same as measured by AUC. Therefore, from the perspective of treatment, different injection routes should not affect the therapeutic effect of the drug. This observation greatly simplifies experimental design for primate toxicity and clinical trials. Example 2 CD24- sialic acid-binding immunoglobulin-like lectin 10 interacts in host response to tissue damage

Nearly two decades ago, Matzinger proposed what is popularly called the danger theory. In essence, she argues that the immune system turns on when it senses danger to the host. Although the dangerous nature cannot be clearly defined in time, it has been determined that necrosis is related to the release of intracellular components such as HMGB1 and heat shock proteins, called DAMP, which is a risk-associated molecular pattern. DAMP was found to promote the production of inflammatory interleukins and autoimmune diseases. In animal models, inhibitors of HMGB1 and HSP90 were found to improve RA. The involvement of DAMP raises the prospect of exploring the negative regulation of host response to DAMP for RA therapy.

Liver necrosis induced by acetaminophen and ensuring inflammation, CD24 was observed to provide strong negative regulation of host responses to tissue damage via interaction with sialic acid-binding immunoglobulin-like lectin G. CD24 is a GPI anchor molecule, which is widely expressed in hematopoietic cells and other tissue stem cells. Genetic analysis of various autoimmune diseases in humans, including multiple sclerosis, systemic lupus erythematosus, RA, and giant cell arthritis, has revealed a significant association between CD24 polymorphisms and the risk of autoimmune diseases. Sialic acid-binding immunoglobulin-like lectin G is a member of the I-lectin family, which is defined by its ability to recognize sialic acid-containing structures. Sialic acid-binding immunoglobulin-like lectin G recognizes sialic acid-containing structures on CD24 and negatively regulates dendritic cells to produce inflammatory interleukins. Human sialic acid-binding immunoglobulin-like lectin 10 and mouse sialic acid-binding immunoglobulin-like lectin G are functionally equivalent in terms of their ability to interact with CD24. However, it is unclear whether there is a one-to-one correlation between mice and human homologs. Although the mechanism remains to be fully elucidated, sialic acid-binding immunoglobulin-like agglutination G-bound SHP1 may be involved in negative regulation. This recent data, reported in Science, has produced a new model in which the CD24-sialic acid-binding immunoglobulin-like lectin G / 10 interaction may distinguish between molecular patterns of pathogen binding (PAMP) and DAMP Play a key role (Figure 4).

At least two overlapping mechanisms can explain the function of CD24. First, by binding to multiple DAMPs, CD24 can retain inflammatory stimuli to prevent them from interacting with TLR or RAGE. This view is supported by the observation that CD24 binds to several DAMP molecules, including HSP70, 90, HMGB1, and nucleolin. Second, perhaps after binding to DAMP, CD24 can stimulate sialic acid-binding immunoglobulin-like lectin G signaling. Both mechanisms work synergistically, as mice with targeted mutations in either gene produce a much stronger inflammatory response. In fact, DCs derived from bone marrow culture of CD24-/-or sialic acid-binding immunoglobulin-like lectin G-/-mice produce much higher inflammatory cytokines when stimulated with HMGB1, HSP70 or HSP90. In contrast, no effect was found in its response to PAMP, such as LPS and PolyI: C. These data not only provide a mechanism by which the innate immune system distinguishes pathogens from tissue damage, but also suggest that CD24 and sialic acid-binding immunoglobulin-like lectin G are potential therapeutic targets for diseases associated with tissue damage. Example 3 CD24 and GvHD prevention

CD24Fc interacts with HMGB1, sialic acid-binding immunoglobulin-like lectin 10 and induces binding between sialic acid-binding immunoglobulin-like lectin G and SHP-1.

To measure the interaction between CD24Fc and sialic acid-binding immunoglobulin-like lectin 10, we fixed CD24Fc to CHIP and used Biacore to measure the binding of sialic acid-binding immunoglobulin-like lectin-10Fc at different concentrations. Happening. As shown in Figure 5A, CD24Fc binds sialic acid-binding immunoglobulin-like lectin 10 with a Kd of 1.6x10 ^-7 M. Its affinity is 100 times higher than the control Fc. The interaction between CD24Fc and HMGB1 was confirmed by a suction experiment using CD24Fc-bound protein G beads, followed by Western blotting using anti-IgG or anti-HMGB1. These data indicate that CD24Fc (but not Fc) binds to HMGB1 and that this binding is cationic dependent (Figure 5B). To determine whether CD24Fc is an agonist of sialic acid-binding immunoglobulin-like lectin G (human counterpart of human sialic acid-binding immunoglobulin-like lectin 10), we use CD24Fc, control Fc, or vehicle (PBS) Controls stimulated CD24-/-splenocytes for 30 minutes. Sialic acid-binding immunoglobulin-like lectin G was then immunoprecipitated and detected with anti-phosphorylated tyrosine or anti-SHP-1. As shown in Figure 5C, CD24Fc induces substantial phosphorylation of sialic acid-binding immunoglobulin-like lectin G and induces binding of SHP-1, a recognized inhibitor of innate immunity the day after tomorrow. Study of in vitro efficacy of CD24Fc.

To study the effect of CD24Fc on the production of inflammatory cytokines by human T cells, mature T cells in human PBML were treated with anti-CD3 antibody (OKT3) (common T cell receptor agonist) in the presence of different concentrations of CD24Fc or human IgG1 Fc )activation. After four days, the supernatant was collected and the production of IFN-γ and TNF-α was measured by enzyme-linked immunosorbent assay (ELISA) to confirm activation. The results in Figure 6 demonstrate that CD24Fc from two different manufacturing batches significantly reduced the production of IFN-γ and TNF-α in activated human PBML compared to the control IgG Fc control. In addition, when CD24Fc was added, cytokine production was inhibited in a dose-dependent manner. Therefore, CD24Fc can inhibit anti-CD3-induced human PBML activation in vitro. This study not only indicates that the mechanism of action of CD24Fc may be inhibited by T cell activation, but also establishes a reliable biological analysis for drug potency and stability testing.

To determine whether CD24Fc regulates the production of inflammatory cytokines in human cell lines, we first silenced CD24 in the human acute mononuclear leukemia THP1 cell line using RNAi, and then induced differentiation into macrophages by treating it with PMA. As shown in FIG. 7A, CD24 silence significantly increased the production of TNFα, IL-1β, and IL-6. These data indicate that endogenous human CD24 plays a major role in limiting the production of inflammatory interleukins. Importantly, CD24Fc recovery inhibited TNFα (Figure 7B) and IL-1β and IL-6 in CD24 silent cell lines. These data not only indicate the relevance of CD24 in the inflammatory response of human cells, but also provide a simple analysis for assessing the biological activity of CD24Fc.

Taken together, these data indicate that CD24Fc can inhibit cytokine production triggered by acquired and innate stimuli. However, because the drug is much more effective at reducing interleukin production by inherent effectors, we believe that the main mechanism of its preventive function is to prevent tissue damage from triggering inflammation early in the transplant. Comparison of therapeutic effects between CsA and CD24Fc in humanized mouse GvHD models

GvHD is known as a major complication of allogeneic BM transplantation. However, GvHD induction in all humanized animal models is dependent on transplantation of a large number of human PBMCs. Some of these humanized animal models do not exhibit the whole-body GvHD seen in humans. Therefore, neonatal NOD / SCID IL2rγ knockout (NSG) mice containing 1.5 million human BM cells were used to develop a humanized whole body GvHD animal model. The results showed that mice exhibited a heterogeneous GvHD with a 100% penetrance and that all mice exhibited high human chimeric traits as early as 14 days after transplantation, which significantly increased to nearly two times after 7 days (data not shown). Depending on the donor used (data not shown), the total mortality rate is 100% within 1-2 months of transplantation. In addition, human T cells infiltrate multiple target organs, including lungs, liver, skin, and intestines. According to the inventors' knowledge, this is the best model for the pathogenesis of acute GVHD in humans, but it is more challenging to treat due to the severity and rapid onset of the disease.

The donors used in both experiments produced abnormally fast and severe GVHD. In order to compare the therapeutic efficacy of cyclosporine A (CsA) and CD24Fc, the maximum tolerable daily dose of CsA (between 0.3 and 1 mg / kg, for up to 4 weeks) in NSG mice after one week of transplantation, This depends on the age of the mice) or with CD24Fc at a dose of 2-4 times (5 mg / kg) per week. As shown in Figure 8, beginning in the second week, rapid onset of GVHD-related deaths in the vehicle group was observed. CD24Fc significantly prolonged the survival of recipient mice (P = 0.015), while CsA did not significantly prolong the survival (P = 0.097). These data indicate that CD24Fc has a therapeutic effect superior to CsA. Example 4 Pharmacokinetics of CD24 in Humans

This example demonstrates the pharmacokinetic analysis of CD24 protein in humans. This was derived from a Phase I, randomized, double-blind, placebo-controlled, single escalating dose study to assess the safety, tolerability, and PK of CD24Fc in healthy male and female adult individuals. A total of 40 individuals were recruited in this study in 5 groups of 8 individuals. Six of the eight individuals in each group received study medication and two individuals received a placebo (0.9% sodium chloride saline). The first group was given 10 mg. Subsequent groups received 30 mg, 60 mg, 120 mg, and 240 mg CD24Fc or matched placebo and were administered at least 3 weeks apart in order to review safety and tolerability data for each previous group. The next higher dose is allowed to be administered to a new group of individuals only if safety and tolerability have been fully demonstrated.

On day 1, one of the initial two individuals in each group was a study drug recipient and one was a placebo recipient. After day 7 (minimum 24 hours between each subgroup), 3rd to 5th individuals and 6th to 8th individuals were administered. Each individual in the same subgroup was administered at least 1 hour apart. If necessary, the rest of the individuals will postpone dosing and review any significant safety issues that may arise in the first or second subgroup of that group during the post-dose period. The latter group was administered at least 3 weeks after the previous group. Screening period:

Screening visits (first visit) up to 21 days before the start of the active treatment period. Subjects provided an informed consent before undergoing an eligible screening procedure. Treatment period:

Subjects were admitted to the Clinical Pharmacology Unit (CPU) on day 1 (second visit), and the randomized treatment period began on day 1 after a minimum of 10 hours of overnight fasting. Individuals were randomly assigned a single dose of CD24Fc or placebo. The subject was detained until the morning of day 4. Follow-up:

All individuals returned to the CPU for follow-up visits on the 7th, 14th, 21st, 28th, and 42th days (± 1) (3rd, 4th, 5th, and 4th visits) 6 visits and 7 visits). The seventh interview was the last interview of all individuals.

Duration of treatment: The total study duration for each individual was up to 63 days. A single dose was administered on the first day.

Number of individuals: Plan: 40 individuals Screening: 224 individuals Random: 40 individuals Complete: 39 individuals Interruption: 1 individual

Diagnosis and main inclusion criteria: The population used in this study was healthy men and women between 18 and 55 years old (including 18 and 55 years old), with a body mass index of 18 kg / m ² and 30 kg / m Between ² (including 18 kg / m ² and 30 kg / m ² ). Research Drugs and Comparative Information:

CD24Fc: A single dose of 10 mg, 30 mg, 60 mg, 120 mg, or 240 mg administered by intravenous infusion; batch number: 09MM-036. CD24Fc is a fully humanized fusion protein, which consists of the mature sequence of human CD24 and the crystallizable fragment region of human immunoglobulin G1 (IgG1Fc). CD24Fc is supplied as a sterile, transparent, colorless, preservative-free aqueous solution for intravenous administration. CD24Fc is formulated as a single-dose injection solution with a concentration of 10 mg / mL and a pH of 7.2. Each CD24Fc vial contains 160 mg of CD24Fc, 5.3 mg of sodium chloride, 32.6 mg of disodium hydrogen phosphate heptahydrate, and 140 mg of sodium dihydrogen phosphate monohydrate in 16 mL ± 0.2 mL of CD24Fc. CD24Fc is supplied in a clear borosilicate glass vial with a chlorobutyl rubber stopper and an easy-to-close aluminum closure.

Matching placebo (0.9% sodium chloride saline) administered via intravenous infusion; batch numbers: P296855, P311852, P300715, P315952.

The willingness to treat (ITT) group consists of all individuals who received at least one dose of the study drug. The ITT group is the main analysis group used for individual information and security assessment.

Clinical laboratory assessments (chemical, hematological, and urinalysis) are summarized based on treatment and consultation. Changes from baseline are also outlined. Vital signs (blood pressure, heart rate, breathing rate, and temperature) are outlined based on treatment and time point. Changes from baseline are also outlined. List all medical information. Overview of ECG parameters and changes from baseline. List general explanations. Plasma CD24Fc concentration

As shown in Figure 9, the average plasma concentration of CD24Fc increased proportionally relative to the administered dose of CD24Fc. For all dose groups except 120 mg, CD24Fc reached its maximum mean plasma concentration 1 hour after administration. For the 120 mg cohort, CD24Fc reached its maximum mean plasma concentration 2 hours after dosing. As of day 42 (984 hours), the average plasma concentration of CD24Fc in all groups had decreased to between 2% and 4% of the maximum average plasma concentration.

Table 1 summarizes the treated plasma CD24Fc PK parameters of the PK evaluable population. Table 1.Summary of pharmacokinetic parameters of plasma CD24Fc in the PK evaluable population Analysis of Plasma CD24Fc Dose Ratio

Figure 10 shows a dose ratio plot of CD24Fc _Cmax versus dose for a PK evaluable population. Figure 11 shows a dose ratio plot of CD24Fc AUC _0-42d versus dose in a PK evaluable population. Figure 12 shows a dose ratio plot of CD24Fc AUC _0-inf versus dose in a PK evaluable population. Table 2 shows the power of dose ratio analysis. Table 2 Power Analysis of Dose Ratio: Plasma CD24Fc Pharmacokinetic Parameters-PK Evaluable Population

The C _max slope is estimated at 1.172 and the 90% CI is 1.105 to 1.240. The AUC _0-42d slope is estimated to be 1.088 and the 90% CI is 1.027 to 1.148. The AUC _0-inf slope is estimated to be 1.087 and the 90% CI is 1.026 to 1.1. Pharmacokinetic conclusions

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Figure 1A shows the amino acid composition of the full-length CD24 fusion protein CD24Fc (also referred to herein as CD24Ig) (SEQ ID NO: 5). The underlined 26 amino acids are signal peptides of CD24 (SEQ ID NO: 4), which divide during secretion from cells expressing the protein and are therefore deleted from the processed form of the protein (SEQ ID NO: 6 )in. The bold portion of the sequence is the extracellular domain of the mature CD24 protein used in the fusion protein (SEQ ID NO: 2). To avoid immunogenicity, the last amino acid (A or V) normally present in mature CD24 proteins has been deleted from the construct. The un-underlined non-bold letters are the sequence of IgG1 Fc, which includes the hinge region and the CH1 and CH2 domains (SEQ ID NO: 7). FIG. 1B shows the sequence of CD24 ^V Fc (SEQ ID NO: 8), wherein the mature human CD24 protein (bold) is a valine polymorphic variant of SEQ ID NO: 1. Figure 1C shows the sequence of CD24 ^A Fc (SEQ ID NO: 9), where the mature human CD24 protein (bold) is an alanine polymorphic variant of SEQ ID NO: 1. The different parts of the fusion protein in Figures 1B and 1C are labeled as in Figure 1A and the variant valine / alanine amino acid is double underlined. Figure 2 shows amino acid changes between a mature CD24 protein (SEQ ID NO: 3) from a mouse and a mature CD24 protein (SEQ ID NO: 2) from a human. Potential O-glycosylation sites are bold and N-glycosylation sites are underlined. Figure 3. WinNonlin compartment modelling analysis of the pharmacokinetics of CD24IgG1 (CD24Fc). Open circles represent the average of 3 mice, and the lines are predicted pharmacokinetic curves. Figure 3A. Intravenous injection of 1 mg CD24IgG1. Figure 3B. Subcutaneous injection of 1 mg of CD24IgG1 (CD24Fc). Figure 3C. Comparison of total antibodies in blood, as measured by area under the curve (AUC), half-life, and maximum blood concentration. It should be noted that, in general, the AUC and Cmax for subcutaneous injections are about 80% of the intravenous injections, but the differences are not statistically significant. Figure 4. Distinguishing CD24-sialic acid-binding immunoglobulin-like lectin (10) (CD24-Siglec G (10)) interactions between PAMP and DAMP. Figure 4A. Host response to PAMP is not affected by CD24-sialic acid binding immunoglobulin-like lectin G (10) interactions. Figure 4B. CD24-sialic acid-binding immunoglobulin-like lectin G (10) interaction inhibits the host's response to DAMP, which may be achieved via SHP-1 which binds to sialic acid-binding immunoglobulin-like lectin G / 10. Figure 5. CD24 Fc binds to sialic acid-binding immunoglobulin-like lectin 10 and HMGB1 and activates sialic acid-binding immunoglobulin-like lectin G (human homolog of human sialic acid-binding immunoglobulin-like lectin 10) . Figure 5A. Affinity measurement for CD24-Fc-sialic acid binding immunoglobulin-like lectin 10 interaction. Figure 5B. CD24-Fc interacts specifically with HMGB-1 in a cation-dependent manner. CD24-Fc was incubated with HMGB1 in 0.1 mM CaCl ₂ and MgCl ₂ in the presence or absence of the cationic chelator EDTA. CD24Fc was attracted by protein G beads, and the amount of HMGB1, CD24Fc or control Fc was determined by Western blot method. Figure 5C. CD24-Fc activates mouse sialic acid-binding immunoglobulin-like lectin G by inducing tyrosine phosphorylation (middle panel) and binding to SHP-1 (top panel). The amount of sialic acid-binding immunoglobulin-like lectin G is shown in the figure below. CD24 ^-/- splenocytes were stimulated with 1 µg / ml CD24-Fc, control Fc or vehicle (PBS) control for 30 minutes. Sialic acid-binding immunoglobulin-like lectin G was then immunoprecipitated and detected with anti-phosphorylated tyrosine or anti-SHP-1. Figure 6. CD24Fc inhibits the production of TNF-α and IFN-γ by anti-CD3 activated human T cells. Human PBML was stimulated with anti-CD3 in the presence or absence of CD24Fc for 4 days, and the amount of IFN-γ and TNF-α released from the cell culture supernatant was measured by ELISA. The data shown are the average of triplicate experiments. Error bars, SEM. Figure 7. CD24 inhibits the production of inflammatory cytokines by human macrophages. Figure 7A. ShRNA silencing of CD24 causes spontaneous production of TNF-α, IL-1β, and IL-6. THP1 cells are transduced with a lentiviral vector encoding promiscuous or two independent CD24 shRNA molecules. Transduced cells were differentiated into macrophages by culturing with PMA (15 ng / ml) for 4 days. After washing off the PMA and non-adherent cells, the cells were cultured for another 24 hours to measure the inflammatory cytokines by the cytokinin bead array. Figure 7B. As in Figure 7A, but with the specified concentration of CD24Fc or control IgG Fc added to the macrophages during the last 24 hours. The data shown in Figure 4A are the mean and standard deviation (SD) of three independent experiments, and Figure 4B represents at least 3 independent experiments. Figure 8. Kaplan-Meier survival analysis of the therapeutic efficacy of CD24Fc and CsA. Summary data from two independent experiments. Figure 9 shows a graph of the treated mean plasma CD24Fc concentration (± SD) of a PK-evaluable population in a human individual. PK = pharmacokinetics; SD = standard deviation. Figure 10 shows a dose ratio plot of CD24Fc _Cmax versus dose for a PK evaluable population. Figure 11 shows a dose ratio plot of CD24Fc AUC _0-42d versus dose in a PK evaluable population. Figure 12 shows a dose ratio plot of CD24Fc AUC _0-inf versus dose in a PK evaluable population.

Claims (40)

A method of treating immune-related adverse events (irAEs) associated with cancer therapy, comprising administering CD24 protein to an individual in need. The method of claim 1, wherein the cancer therapy is an anti-CTLA4 antibody. The method according to item 2 of the patent application, wherein the anti-CTLA4 antibody is Ipilimumab. The method of claim 2 in which the anti-CTLA4 antibody is administered in combination with another therapy. The method of claim 1, wherein the cancer therapy is an anti-PD-1 antibody. The method of claim 5 in which the anti-PD-1 antibody is administered in combination with another therapy. The method of claim 1, wherein the cancer therapy is an anti-PD-L1 antibody. The method of claim 7 in which the anti-PD-L1 antibody is administered in combination with another therapy. The method of claim 1, wherein the cancer therapy is a chimeric antigen receptor T cell. The method of claim 1, wherein the cancer therapy is a T cell modified by a T cell receptor (TCR). The method of claim 1, wherein the cancer therapy is activating natural killer cells. The method of claim 1, wherein the cancer therapy is radiation therapy. The method of claim 1, wherein the cancer therapy is chemotherapy. The method of claim 1, wherein the cancer therapy involves an antibody that targets cancer cells. The method of claim 1, wherein the CD24 protein comprises mature human CD24 or a variant thereof. The method of claim 15, wherein the mature human CD24 comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2. The method of claim 1, wherein the CD24 protein is soluble. The method of claim 1, wherein the CD24 protein is glycosylated. The method of claim 15, wherein the CD24 protein further comprises a protein tag, wherein the protein tag is fused at the N-terminus or C-terminus of the CD24 protein. The method of claim 19, wherein the protein tag comprises a portion of mammalian immunoglobulin (Ig). The method of claim 20, wherein the Ig portion is an Fc region of a human Ig protein. The method of claim 21, wherein the Fc region comprises a hinge region and CH2 and CH3 domains of the human Ig protein, and wherein the Ig is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and IgA. For example, the method of claim 21, wherein the Fc region comprises a hinge region of IgM and CH2, CH3, and CH4 domains. The method of claim 22, wherein the sequence of the CD24 protein comprises the amino acid sequence set forth in SEQ ID NO: 6, 11, or 12. The method of claim 1, wherein the CD24 protein is produced using a eukaryotic protein expression system. The method of claim 25, wherein the expression system comprises a vector or a replication-deficient retroviral vector contained in a Chinese hamster ovary cell line. The method of claim 26, wherein the replication-deficient retroviral vector is stably integrated into the genome of a eukaryotic cell. The method of claim 1, wherein the irAE is diarrhea or another gastrointestinal disorder. For example, the method of claim 1, wherein the irAE is pure red blood cell hypoplasia. The method according to item 1 of the patent application, wherein the irAE is microerythrocytic anemia. The method of claim 1, wherein the irAE is lupus. According to the method of claim 1, wherein the irAE is autoimmune nephritis. According to the method of claim 1, wherein the irAE is autoimmune hepatitis. The method according to item 1 of the patent application, wherein the irAE is pneumonia. The method of claim 1, wherein the irAE is heart disease, such as myocarditis and pericarditis. According to the method of claim 1, wherein the irAE is an endocrine lesion. The method of claim 1, wherein the irAE is Addison's disease. For example, the method of claim 1, wherein the irAE is hypogonadism. For example, the method of claim 1, wherein the irAE is Sjogren's syndrome. For example, the method of claim 1, wherein the irAE is type I diabetes.